A variety of approaches has been used to improve the economy of biologically-based industrial processes by "improving" the organism involved. These techniques constitute what may be categorized as strain improvement programs. The efficacy of improving said processes is dependent on the type of organism and the nature of the end-product.
The success of any strain improvement program will be directly affected by the facility with which genetic diversity can be generated in the subject organism, or alternatively the ease with which the genetic diversity already present in nature can be evaluated.
A colony that appears on agar medium following plating out of spores, cells, or small hyphal fragments consists of a population most of which are genetically identical, although some cells may differ due to spontaneous mutation during the growth of the colony or to nuclear heterogeneity in the original propagule.
It was the rare occurrence of spontaneous mutations within existing cultures that provided the major source of strain improvement germplasm in the early years of the fermentation industry. A secondary source of improved strains was nature itself, that is, the isolation from nature of previously unknown strains with improved characteristics.
As a fuller understanding of the biological and chemical basis of genetic change developed, strain improvement programs incorporated this new knowledge into their rationale. For example, induced mutagenesis to generate genetic diversity followed by the subsequent screening, selection and purification of superior strains represents one of the most effective means of improving the yield of a fermentation product. Mutation programs are vital to the fermentation industry in that higher productivities exhibited by the new strains are essential in reducing costs.
It is now appreciated that the choice of a particular mutagen as well as the actual conditions of mutagenesis can play a major role in determining the types and numbers of mutants recovered during a strain improvement program. In general, two experimental approaches have been used to recover new strains resulting from induced mutagenesis experiments; these are: screening and selection.
In a screening system all strains grow with the exception of those killed outright as a result of the mutagenesis treatment; thus each isolate must be examined to identify the desired characteristic. Since tens of millions of isolates must be examined, this approach can be highly labor intensive.
In a selection system, the experimental conditions are chosen so as to establish a growth differential between the rare strains possessing the desired characteristic and all other strains which do not possess said trait. In certain instances the selected strain will not grow under the conditions of the experiment while the non-selected strains will grow. Thus, by removing the growing strains by filtration or other means, the size of the population of cells remaining to be examined is dramatically reduced. Alternatively, conditions may be established such that the selected strain will grow while the non-selected strains are inhibited, here again effectively reducing the population to be examined.
Although induced mutagenesis has been an extremely powerful force in the area of strain improvement, there are some limitations. For example, as more and more mutations are accumulated in a strain as a result of the continuing improvement program, a saturation level is reached. Subjecting such a strain to further selection often results in a loss of productivity due to reversion of existing mutations.
A more fundamental limtation exists in induced-mutation based improvement programs, namely, such programs are based on the assumption that the strains possess the activity to be improved. In other words, the organism must possess, in its genetic repertoire, the information to direct the synthesis of a gene product before any genetically-based improvement program relating to the function of the product may be considered.
A variety of genetic approaches has been developed to reduce these limitations. For example, hybridization techniques allow for genetic recombination to occur among a number of different strains. Hybridization can be achieved by means of sexual reproduction or asexual processes such as somatic cell fusion or heterokaryon formation. The advent of recombinant DNA technology has reduced the limitations on improvement programs even further. The ability to transfer genes between organisms of widely divergent genetic backgrounds has provided the experimenter with a virtually limitless supply of genetic information upon which to improve. This advent of genetic engineering technology has prompted a renewed interest, natural sources of genetic variability, not with a view toward isolating and developing new strains, per se, but rather as a source of as little as a single gene which may be transferred to already established strains.
Regardless of the source of the variant strain, be it either nature, a spontaneous mutation, an induced mutation, or a recombinant resulting from sexual, asexual or genetic engineering processes, methods of screening and selection remain of critical importance, allowing the experimenter to recover the variant strain from among the population of existing strains from which it arose.
In light of the subject invention, one group of organisms of particular interest with regard to strain improvement programs are those useful as sources of amylolytic (starch degrading) enzymes. These enzymes fall into two main classes based upon the linkages characteristic of the substrates upon which the enzymes act. One class, the .alpha.-1,4-glucanases contain enzymes which degrade glucose polymers having .alpha.-1,4-linkages and do so by either randomly cleavaging at points within the polymer chain, that is they are endo-.alpha.-1,4-glucanases or degrade the polymer from the terminus, those being characterized as exo-.alpha.-1,4-glucanases. The endo-.alpha.-1,4-glucanase type includes enzymes such as .alpha.-amylase, where as the exo-.alpha.-1,.alpha.4-glucanase type include enzymes such as exo-maltohexohydrolase, .beta.-amylase and amyloglucosidase.
The second class of starch degrading enzymes are the .alpha.-1,6-glucanases, the so-called "debranching enzymes" owing to their affinity for degrading at linkages characterizing the branch points of starch molecules. Both endo (pullulanase and isoamylase) and exo-(exo-pullulanase) forms are known.
Bacterial .alpha.-amylase (.alpha.,-1,4-glucan-glucanohydrolase, E.C. 3.2.1.1) acts, as an endolase, on starch components which contain a minimum of three linked glucose units, resulting in the formation of reducing sugars. These enzymes are useful in desizing textile fabrics, in modifying starches suitable for preparation of adhesives, sizes and coatings for the paper industry, as well as in the manufacturing of glucose, and glucose syrups. Fungal .alpha.-amylases are extensively used in flour treatment processes.
Amylases have been prepared from microbiological cultures of Bacillus. In British Specification No. 1,296,839, there is described a process for producing thermally stable alpha amylase by the cultivation of Bacillus licheniformis. The enzyme so produced is of significantly higher thermal stability than the .alpha.-amylase produced by Bacillus subtilis. However yields and activity of the enzymes recovered leave much to be desired as far as commercial production is concerned.
The disclosure by Horwath, (copending and cofiled U.S. application Ser. No. 480,428) which is incorporated herein by reference, of a new strain of Bacillus licheniformis particularly useful for commercial production has warranted the development of a large scale, efficient, combination selection and screening system for the recovery of .alpha.-amylase producing strains of microorganisms. It is the principle object of the instant invention to provide such a combination selection and screening system.